Mass spectrometric imaging is a technique for investigating the distribution of a substance having a specific mass-to-charge ratio (m/z) by performing a mass analysis on each of a plurality of micro areas within a two-dimensional area of a sample, such as a piece of living tissue. This technique is expected to be applied, for example, in drug discovery, biomarker discovery, and investigation on the causes of various diseases. Mass spectrometers designed for mass spectrometric imaging are generally referred to as imaging mass spectrometers. This device may also be called a mass microscope since its operation normally includes performing a microscopic observation of an arbitrary area on the sample, selecting a region of interest based on the microscopically observed image, and performing a mass analysis of the selected region. For example, the configurations of commonly known mass microscopes and analysis examples obtained those mass microscopes are disclosed in International Publication No. WO 2008/068847; Kiyoshi OGAWA et al., “Kenbi Shitsuryou Bunseki Souchi No Kaihatsu (Research and Development of Mass Microscope)”, Shimadzu Hyouron (Shimadzu Review), Vol. 62, No. 3/4, pp. 125-135, Mar. 31, 2006; and Harada et al. “Kenbi Shitsuryou Bunseki Souchi Ni Yoru Seitai Soshiki Bunseki (Biological Tissue Analysis using Mass Microscope”, Shimadzu Hyouron (Shimadzu Review), Vol. 64. No. 3/4, pp. 139-145, Apr. 24, 2008.
A mass microscope is basically composed of a microscopic observation means for performing a microscopic observation of a two-dimensional area on a sample and a mass analysis means for performing a mass analysis for each of a plurality of portions within the two-dimensional area on the sample. The microscopic observation means can be divided into two major types: One type has an imaging means (e.g. a CCD camera) and a display unit (e.g. a monitor) with a screen on which an image taken with the imaging means can be displayed, thus allowing an operator to observe a sample image; the other type is a normal microscope having an eyepiece. The mass analysis means includes an ionization means for ionizing a component contained in a sample, an ion separation/detection means for separating the ions originating from the sample according to their mass-to-charge ratio and detecting each ion, and an ion transport means for guiding and transporting the ions generated from the sample to the ion-separating/detecting means. The microscopic observation means and the mass analysis means are not always provided in the same system; they can each be configured as a separate unit.
The primary subjects of analysis by the mass microscope are biological samples. Biological samples easily suffer from damage when irradiated with laser light. Accordingly, a matrix assisted laser desorption ion source (MALDI ion source) is normally used to ionize this type of sample. When the sample is a tissue section, the sample is in the form of an extremely thin slice (with a thickness of a few micrometers to several tens of micrometers) placed on a sample plate, on which a matrix solution is applied by an appropriate method, such as spraying or coating. In any application method, the sample surface is covered with a crystallized matrix after the solution is dried. Therefore, in many cases, the observed image of the sample becomes rather obscure.
When the region of interest for the mass spectroscopic imaging is selected on such an obscured sample image taken after the application of the matrix, it is difficult to correctly select the intended region. To accurately and properly perform the mass spectroscopic imaging, the target region must be determined based on a clear sample image taken before the application of the matrix. Accordingly, a procedure for mass spectroscopic imaging normally includes the following successive steps: a sample plate, with a sample placed thereon, is set in a mass spectrometer; an image of this sample is taken and saved as a sample image before matrix application; the sample plate is temporarily removed from the apparatus; a matrix is applied to the sample surface; the sample plate is re-set in the apparatus; and a mass analysis is performed on a region determined with reference to the sample image taken before the matrix application.
When being re-set in the apparatus, the sample plate may be set at a position displaced from the position where it was before its removal. If this occurs, the actual area of analysis will be displaced from the target region that has been selected with reference to the sample image taken before the application of the matrix. Such a displacement in the position of the re-set sample plate is much larger than the spatial resolution of the mass microscope, which is capable of performing the mass spectroscopic imaging with a spatial resolution of equal to or less than several tens of micrometers. Therefore, the aforementioned displacement poses a serious problem for accurately performing the mass spectroscopic imaging.
In the case where the microscopic observation means is configured as a separate microscope, the image of the sample placed on the sample plate, taken with the microscope, is initially saved in a memory of the microscope and subsequently read out by the mass spectrometer. After the sample plate is removed from the microscope and the matrix is applied on the sample surface, the sample plate is re-set in the mass spectrometer. The mass spectrometer performs the mass analysis on a region determined based on the microscopic image of the sample.
In this system, the position of the sample plate set in the mass spectrometer may be displaced from the position where the microscopic image of the sample plate was taken. If this occurs, the actual area of analysis will be displaced from the target region selected based on the sample image taken before the application of the matrix.
One method aimed at solving the aforementioned problem is disclosed in “flexControl User Manual”, First Edition, Bruker Daltonics, Bremen, Germany, 2006, pp. 3-35. According to this method, before taking a microscopic image, an operator puts a mark for position recognition on the sample plate with a pen or the like. After setting the sample plate in the mass spectrometer, the operator locates the position-recognition mark on the sample plate through an imaging device annexed to the mass spectrometer and indicates the position of the mark. The position of this mark thus observed on the sample plate set in the apparatus is subsequently used as a reference point for controlling the position of the sample stage so that the measurement range selected on the microscopic image will be analyzed.
However, the mark that is manually put on the sample plate by the operator inevitably becomes large. Furthermore, the process of locating the mark on the sample plate set in the mass spectrometer uses a low-resolution image produced without using the microscope. The use of a large mark and a low-resolution image makes it difficult to improve the positioning accuracy.
In a mass spectrometer disclosed in WO2008/068847, which is configured as a single apparatus having a microscope and a mass analysis unit, a marker for position identification is originally provided on a sample plate. The magnitude and direction of the displacement of the sample plate between the first position where the sample plate was initially set and the second position where the sample plate is located after being re-set in the apparatus is calculated by comparing two images taken when the sample plate was at the first and second positions, respectively, During the analysis, the position of the sample stage is controlled so as to cancel the calculated displacement. The aforementioned document also discloses a technique for calculating the magnitude and direction of the displacement by means of a specific pattern or color that can be identified even after the application of the matrix.
Creating a sample plate with a marker for position identification requires special machining/processing work, which makes the sample plate more expensive and increases the operating cost of the analysis. Furthermore, comparing a portion of the sample images before and after the application of the matrix does not always provide satisfactorily accurate information about the displacement since this method is affected by the state of the applied matrix and the condition of the sample. For these reasons, it is desired to develop a method in which a conventional sample plate that requires no special work can be used, and in which the displacement of the sample plate can be accurately detected and cancelled by a technique different from the method of comparing sample images taken before and after the application of the matrix.
In some cases, such as an analysis of a set of samples prepared by consecutively slicing the same biological tissue, the prepared samples are extremely similar to each other in shape, pattern and color and hence difficult to be visually distinguished. As a result, one sample may be mistaken for another sample when the analysis is performed or the samples are put into storage. A method for preventing this problem has been desired.
After a sample plate carrying a sample with a matrix applied thereto is re-set in the apparatus, when the analysis is performed, it is necessary to retrieve from the storage device the sample image taken before the application of the matrix and determine the area of analysis. Searching for the sample image concerned consumes considerable time and labor if there are an enormous number of samples to be sequentially analyzed. This problem can be avoided by repeating the analyzing work for each sample. However, this method considerably deteriorates the throughput of the analysis since applying and drying a matrix normally requires a certain period of time.
The present invention has been developed in view of the previously described problems. Its first objective is to provide a mass spectrometer that allows the use of an inexpensive sample plate which requires no special processing, and yet can correctly detect and cancel the displacement of the sample plate resulting from its removal from and re-setting in the apparatus so as to perform the mass spectroscopic imaging on the intended area.
The second objective of the present invention is to provide a mass spectrometer capable of correctly identifying each sample and subjecting it to analysis even if there are a large number of samples having similar appearances.
The third objective of the present invention is to provide a mass spectrometer capable of quickly and correctly retrieving sample images taken before the application of the matrix and determining the area of analysis even in the case of analyzing a large number of samples.